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3. Gram-positive anaerobic cocci guard skin homeostasis by regulating host-defense mechanisms.

Author

Krieken, D.A. van der, Rikken, G., Ederveen, T.H.A., Jansen, P.A.M., Rodijk-Olthuis, D., Meesters, L.D., Vlijmen-Willems, I.M.J.J. van, Cranenbroek, B. van, Molen, R.G. van der, Schalkwijk, J., Bogaard, E.H.J. van den, Zeeuwen, P.L.J.M., Krieken, D.A. van der, Rikken, G., Ederveen, T.H.A., Jansen, P.A.M., Rodijk-Olthuis, D., Meesters, L.D., Vlijmen-Willems, I.M.J.J. van, Cranenbroek, B. van, Molen, R.G. van der, Schalkwijk, J., Bogaard, E.H.J. van den, and Zeeuwen, P.L.J.M.

Abstract

Item does not contain fulltext, In atopic dermatitis (AD), chronic skin inflammation is associated with skin barrier defects and skin microbiome dysbiosis including a lower abundance of Gram-positive anaerobic cocci (GPACs). We here report that, through secreted soluble factors, GPAC rapidly and directly induced epidermal host-defense molecules in cultured human keratinocytes and indirectly via immune-cell activation and cytokines derived thereof. Host-derived antimicrobial peptides known to limit the growth of Staphylococcus aureus-a skin pathogen involved in AD pathology-were strongly upregulated by GPAC-induced signaling through aryl hydrocarbon receptor (AHR)-independent mechanisms, with a concomitant AHR-dependent induction of epidermal differentiation genes and control of pro-inflammatory gene expression in organotypic human epidermis. By these modes of operandi, GPAC may act as an "alarm signal" and protect the skin from pathogenic colonization and infection in the event of skin barrier disruption. Fostering growth or survival of GPAC may be starting point for microbiome-targeted therapeutics in AD.

Published
2023

4. CYSRT1: an antimicrobial epidermal protein that can interact with late cornified envelope proteins

Author

Niehues, H., Rikken, G., Kersten, F.F.J., Eeftens, J.M., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Jansen, P.A.M., Hendriks, W.J.A.J., Ederveen, T.H., Schalkwijk, J., Bogaard, E.H.J. van den, Zeeuwen, P.L.J.M., Niehues, H., Rikken, G., Kersten, F.F.J., Eeftens, J.M., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Jansen, P.A.M., Hendriks, W.J.A.J., Ederveen, T.H., Schalkwijk, J., Bogaard, E.H.J. van den, and Zeeuwen, P.L.J.M.

Abstract

Contains fulltext : 294910.pdf (Publisher’s version ) (Open Access)

Published
2023

5. Novel methodologies for host-microbe interactions and microbiome-targeted therapeutics in 3D organotypic skin models

Author

Rikken, Gijs, Meesters, L.D., Jansen, P.A.M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Niehues, H., Schalkwijk, J., Zeeuwen, P.L.J.M., Bogaard, E.H.J. van den, Rikken, Gijs, Meesters, L.D., Jansen, P.A.M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Niehues, H., Schalkwijk, J., Zeeuwen, P.L.J.M., and Bogaard, E.H.J. van den

Abstract

Item does not contain fulltext

Published
2023

13. Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures

Author

Niehues, H., Jansen, P.A.M., Rodijk-Olthuis, D., Rikken, G., Smits, J.P.H., Schalkwijk, J., Zeeuwen, P.L.J.M., Bogaard, E.H.J. van den, Niehues, H., Jansen, P.A.M., Rodijk-Olthuis, D., Rikken, G., Smits, J.P.H., Schalkwijk, J., Zeeuwen, P.L.J.M., and Bogaard, E.H.J. van den

Abstract

Contains fulltext : 229256.pdf (Publisher’s version ) (Open Access), In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.

Published
2020

15. Psoriasis-Associated Late Cornified Envelope (LCE) Proteins Have Antibacterial Activity

Author

Niehues, H., Tsoi, L.C., Krieken, D.A. van der, Jansen, P.A.M., Oortveld, M.A.W., Rodijk-Olthuis, D., Vlijmen, I.M.J.J. van, Hendriks, W.J.A.J., Helder, R.W.J., Bouwstra, J.A., Bogaard, E.H. van den, Stuart, P.E., Nair, R.P., Elder, J.T., Zeeuwen, P.L.J.M., Schalkwijk, J., Krieken, D. A. van der, Oortveld, M.A.W, Krieken, D. A. van der, and Oortveld, M.A.W

Subjects
Keratinocytes, Male, 0301 basic medicine, Genotype, Human skin, Dermatology, Quantitative trait locus, Biology, Real-Time Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Biochemistry, Linkage Disequilibrium, Article, Cornified envelope, 03 medical and health sciences, Cornified Envelope Proline-Rich Proteins, Psoriasis, medicine, Stratum corneum, Humans, Life Science, Genetic Predisposition to Disease, Molecular Biology, Gene, Cells, Cultured, integumentary system, Biopsy, Needle, Other Research Radboud Institute for Health Sciences [Radboudumc 0], Role, Wild type, Cell Biology, medicine.disease, Immunohistochemistry, Immunity, Innate, Anti-Bacterial Agents, Cell biology, 030104 developmental biology, medicine.anatomical_structure, Gene Expression Regulation, Immunology, Female, Nanomedicine Radboud Institute for Molecular Life Sciences [Radboudumc 19], Function (biology), Inflammatory diseases Radboud Institute for Molecular Life Sciences [Radboudumc 5]
Abstract

Contains fulltext : 182646.pdf (Publisher’s version ) (Open Access) Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.

Published
2017

16. STAT1 gain-of-function compromises skin host defense in the context of IFN-gamma signaling

Author

Niehues, H., Rosler, B., Krieken, D.A. van der, Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Peppelman, M., Schalkwijk, J., Bogaard, E.H.J. van den, Zeeuwen, P.L.J.M., Veerdonk, F.L. van de, Niehues, H., Rosler, B., Krieken, D.A. van der, Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Peppelman, M., Schalkwijk, J., Bogaard, E.H.J. van den, Zeeuwen, P.L.J.M., and Veerdonk, F.L. van de

Abstract

Contains fulltext : 205190.pdf (publisher's version ) (Open Access)

Published
2019

19. Gram-positive anaerobe cocci are underrepresented in the microbiome of filaggrin-deficient human skin

Author

Zeeuwen, P.L.J.M., Ederveen, T.H., Krieken, D.A. van der, Niehues, H., Boekhorst, J., Kezic, S., Hanssen, D.A., Otero, M.E., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Falcone, D., Bogaard, E.H.J. van den, Kamsteeg, M., Koning, H.D. de, Zeeuwen-Franssen, M.E., Steensel, M.A. van, Kleerebezem, M., Timmerman, H.M., Hijum, S.A.F.T. van, Schalkwijk, J., Zeeuwen, P.L.J.M., Ederveen, T.H., Krieken, D.A. van der, Niehues, H., Boekhorst, J., Kezic, S., Hanssen, D.A., Otero, M.E., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Falcone, D., Bogaard, E.H.J. van den, Kamsteeg, M., Koning, H.D. de, Zeeuwen-Franssen, M.E., Steensel, M.A. van, Kleerebezem, M., Timmerman, H.M., Hijum, S.A.F.T. van, and Schalkwijk, J.

Abstract

Contains fulltext : 174203.pdf (publisher's version ) (Closed access)

Published
2017

21. Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier function

Author

Niehues, H., Schalkwijk, J., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Rossum, M.M. van, Wladykowski, E., Brandner, J.M., Bogaard, E.H.J. van den, Zeeuwen, P.L.J.M., Niehues, H., Schalkwijk, J., Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Rossum, M.M. van, Wladykowski, E., Brandner, J.M., Bogaard, E.H.J. van den, and Zeeuwen, P.L.J.M.

Abstract

Contains fulltext : 174196.pdf (publisher's version ) (Closed access)

Published
2017

22. Polymorphisms inCD84,IL12BandTNFAIP3are associated with response to biologics in patients with psoriasis

Author

van den Reek, J.M.P.A., primary, Coenen, M.J.H., additional, van de L'Isle Arias, M., additional, Zweegers, J., additional, Rodijk-Olthuis, D., additional, Schalkwijk, J., additional, Vermeulen, S.H., additional, Joosten, I., additional, van de Kerkhof, P.C.M., additional, Seyger, M.M.B., additional, Zeeuwen, P.L.J.M., additional, and de Jong, E.M.G.J., additional

Published
2017
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26. Mast-cell interleukin-1beta, neutrophil interleukin-17 and epidermal antimicrobial proteins in the neutrophilic urticarial dermatosis in Schnitzler's syndrome

Author

Koning, H.D. de, Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Meer, J.W.M. van der, Zeeuwen, P.L.J.M., Simon, A., Schalkwijk, J., Koning, H.D. de, Vlijmen-Willems, I.M.J.J. van, Rodijk-Olthuis, D., Meer, J.W.M. van der, Zeeuwen, P.L.J.M., Simon, A., and Schalkwijk, J.

Abstract

Contains fulltext : 153351.pdf (publisher's version ) (Closed access), BACKGROUND: Schnitzler's syndrome (SchS) is an autoinflammatory disease characterized by a chronic urticarial rash, a monoclonal component and signs of systemic inflammation. Interleukin (IL)-1beta is pivotal in the pathophysiology. OBJECTIVES: Here we investigated the cellular source of proinflammatory mediators in the skin of patients with SchS. METHODS: Skin biopsies of lesional and nonlesional skin from eight patients with SchS and healthy controls, and patients with cryopyrin-associated periodic syndrome (CAPS), delayed-pressure urticaria (DPU) and cold-contact urticaria (CCU) were studied. We studied in vivoIL-1beta, IL-17 and antimicrobial protein (AMP) expression in resident skin cells and infiltrating cells. In addition we investigated the in vitro effect of IL-1beta, IL-17 and polyinosinic-polycytidylic acid (poly:IC) stimulation on cultured epidermal keratinocytes. RESULTS: Remarkably, we found IL-1beta-positive dermal mast cells in both lesional and nonlesional skin of patients with SchS, but not in healthy control skin and CCU, and fewer in CAPS. IL-17-positive neutrophils were observed only in lesional SchS and DPU skin. In lesional SchS epidermis, mRNA and protein expression levels of AMPs were strongly increased compared with nonlesional skin and that of healthy controls. When exposed to IL-1beta, poly:IC or IL-17, patient and control primary human keratinocytes produced AMPs in similar amounts. CONCLUSIONS: Dermal mast cells of patients with SchS produce IL-1beta. This presumably leads to activation of keratinocytes and neutrophil influx, and further amplification of inflammation by IL-17 (from neutrophils and mast cells) and epidermal AMP production leading to chronic histamine-independent neutrophilic urticarial dermatosis.

Published
2015

28. Polymorphisms in CD84, IL12B and TNFAIP3 are associated with response to biologics in patients with psoriasis.

Author

Reek, J.M.P.A., Coenen, M.J.H., L'Isle Arias, M., Zweegers, J., Rodijk ‐ Olthuis, D., Schalkwijk, J., Vermeulen, S.H., Joosten, I., Kerkhof, P.C.M., Seyger, M.M.B., Zeeuwen, P.L.J.M., and Jong, E.M.G.J.

Subjects
PSORIASIS treatment, BIOTHERAPY, HETEROGENEITY, ETANERCEPT, RHEUMATOID arthritis, THERAPEUTICS
Abstract

Background The effectiveness of biologics for psoriasis shows heterogeneity among patients. With pharmacogenetic markers, it might be possible to predict treatment response. Objectives We aimed to test the association between genetic markers and the response to biologics in psoriasis (etanercept, adalimumab, ustekinumab) in a prospective cohort. Methods We investigated the copy number variation in the LCE3B and LCE3C genes, and eight single-nucleotide polymorphisms ( SNPs) in HLA-C*06, CD84, IL12B, IL23R, TRAF3 IP2, ERAP1, IFIH1 and TNFAIP3. The decrease in Psoriasis Area and Severity Index ( PASI) was calculated as ∆ PASI (absolute PASI decrease compared with baseline) and PASI 75 (proportion of patients with ≥ 75% improvement vs. baseline). Associations between genetic variants and treatment outcome were assessed using multivariable linear regression analysis (∆ PASI corrected for baseline PASI, primary analysis) and Pearson's χ2-test or Fisher's exact test ( PASI 75, secondary analysis). Results We included 348 treatment episodes in 234 patients. Patients heterozygous ( GA) for the SNP in CD84 (rs6427528) had a better ∆ PASI response to etanercept after 3 months ( P = 0·025) than the homozygous reference group ( GG). In addition, patients heterozygous ( CT) for the IL12B variant showed a better response (∆ PASI) to ustekinumab ( P = 0·017) than the reference group ( CC). Patients homozygous ( GG) for the SNP in TNFAIP3 showed a worse response (∆ PASI) to ustekinumab ( P = 0·031) than the reference group ( TT). The associations with ustekinumab resulting from the primary analysis were not confirmed in the secondary ( PASI 75) analysis. Conclusions We demonstrated a strong association between etanercept use in psoriasis and variations in CD84, a gene that was previously found to be a predictor of response to etanercept in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Published
2017
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29. Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

Author

Bogaard, E.H.J. van den, Rodijk-Olthuis, D., Jansen, P.A.M., Vlijmen-Willems, I.M.J.J. van, Erp, P.E.J. van, Joosten, I., Zeeuwen, P.L.J.M., Schalkwijk, J., Bogaard, E.H.J. van den, Rodijk-Olthuis, D., Jansen, P.A.M., Vlijmen-Willems, I.M.J.J. van, Erp, P.E.J. van, Joosten, I., Zeeuwen, P.L.J.M., and Schalkwijk, J.

Abstract

1 september 2012, Contains fulltext : 109407.pdf (publisher's version ) (Closed access), The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.

Published
2012

30. Strong induction of AIM2 expression in human epidermis in acute and chronic inflammatory skin conditions

Author

de Koning, H.D., Bergboer, J.G.M., Bogaard, E.H.J. van den, van Vlijmen-Willems, I.M., Rodijk-Olthuis, D., Simon, A., Zeeuwen, P.L.J.M., Schalkwijk, J., de Koning, H.D., Bergboer, J.G.M., Bogaard, E.H.J. van den, van Vlijmen-Willems, I.M., Rodijk-Olthuis, D., Simon, A., Zeeuwen, P.L.J.M., and Schalkwijk, J.

Abstract

Contains fulltext : 109608.pdf (publisher's version ) (Closed access), Absent in melanoma 2 (AIM2) is a double-stranded DNA receptor, and its activation initiates an interleukin-1 beta processing inflammasome. AIM2 is implicated in host defense against several pathogens, but could hypothetically also contribute to autoinflammatory or autoimmune diseases, such as is the case for NLRP3. Using thoroughly characterised antibodies, we analysed AIM2 expression in human tissues and primary cells. A strong epidermal upregulation of AIM2 protein expression was observed in several acute and chronic inflammatory skin disorders, such as psoriasis, atopic dermatitis, venous ulcera, contact dermatitis, and experimental wounds. We also found AIM2 induction by interferon-gamma in submerged and three-dimensional in vitro models of human epidermis. Our data highlight the dynamics of epidermal AIM2 expression, showing Langerhans cell and melanocyte-restricted expression in normal epidermis but a pronounced induction in subpopulations of epidermal keratinocytes under inflammatory conditions.

Published
2012

31. Epidermal expression of host response genes upon skin barrier disruption in normal skin and uninvolved skin of psoriasis and atopic dermatitis patients

Author

de Koning, H.D., Kamsteeg, M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Erp, P.E.J. van, Schalkwijk, J., Zeeuwen, P.L.J.M., de Koning, H.D., Kamsteeg, M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Erp, P.E.J. van, Schalkwijk, J., and Zeeuwen, P.L.J.M.

Abstract

Contains fulltext : 95864.pdf (publisher's version ) (Closed access)

Published
2011

32. Psoriasis risk genes of the late cornified envelope-3 group are distinctly expressed compared with genes of other LCE groups

Author

Bergboer, J.G.M., Tjabringa, G.S., Kamsteeg, M., Vlijmen-Willems, I.M. van, Rodijk-Olthuis, D., Jansen, P.A.M., Thuret, J.Y., Narita, M., Ishida-Yamamoto, A., Zeeuwen, P.L.J.M., Schalkwijk, J., Bergboer, J.G.M., Tjabringa, G.S., Kamsteeg, M., Vlijmen-Willems, I.M. van, Rodijk-Olthuis, D., Jansen, P.A.M., Thuret, J.Y., Narita, M., Ishida-Yamamoto, A., Zeeuwen, P.L.J.M., and Schalkwijk, J.

Abstract

Contains fulltext : 98267.pdf (publisher's version ) (Closed access), Deletion of the late cornified envelope (LCE) genes LCE3B and LCE3C has recently been identified as a risk factor for psoriasis. Expression of 16 LCE genes of LCE groups 1, 2, 3, 5, and 6 was examined in vivo and in vitro. Quantitative PCR demonstrated that moderate to high LCE expression was largely confined to skin and a few oropharyngeal tissues. Genes of the LCE3 group demonstrated increased expression in lesional psoriatic epidermis and were induced after superficial injury of normal skin, whereas expression of members of other LCE groups was down-regulated under these conditions. Immunohistochemistry and immunoelectron microscopy demonstrated that LCE2 protein expression was restricted to the uppermost granular layer and the stratum corneum. Stimulation of in vitro reconstructed skin by several psoriasis-associated cytokines resulted in induction of LCE3 members. The data suggest that LCE proteins of groups 1, 2, 5, and 6 are involved in normal skin barrier function, whereas LCE3 genes encode proteins involved in barrier repair after injury or inflammation. These findings may provide clues to the mechanistic role of LCE3B/C deletion in psoriasis.

Published
2011

33. The cystatin M/E-cathepsin L balance is essential for tissue homeostasis in epidermis, hair follicles, and cornea.

Author

Zeeuwen, P.L.J.M., Vlijmen-Willems, I.M.J.J. van, Cheng, T., Rodijk-Olthuis, D., Hitomi, K., Hara-Nishimura, I., John, S., Smyth, N., Reinheckel, T., Hendriks, W.J.A.J., Schalkwijk, J., Zeeuwen, P.L.J.M., Vlijmen-Willems, I.M.J.J. van, Cheng, T., Rodijk-Olthuis, D., Hitomi, K., Hara-Nishimura, I., John, S., Smyth, N., Reinheckel, T., Hendriks, W.J.A.J., and Schalkwijk, J.

Abstract

1 oktober 2010, Contains fulltext : 88970.pdf (publisher's version ) (Closed access), Cystatin M/E (CST6) is a nonredundant, epithelium-specific protease inhibitor with a presumed role in epidermal differentiation and tumor suppression. We have previously reported that cystatin M/E deficiency in Cst6(-/-) mice causes neonatal lethality because of excessive transepidermal water loss. Biochemical evidence suggests that cystatin M/E controls the activity of legumain, cathepsin L, cathepsin V, and transglutaminase-3. Using a genetic approach we sought to define the role of cystatin M/E in epithelial biology by identification of its target proteases and their downstream functions. Ablation of cathepsin L in a Cst6(-/-) background (Cst6(-/-)Ctsl(-/-) double-knockout mice) restored viability and resulted in normalization of stratum corneum morphology. Ablation of legumain or transglutaminase-3 in Cst6(-/-) mice, however, did not rescue the lethal phenotype. Intriguingly, both Cst6(-/-)Ctsl(-/-) and Cst6(-/-)Ctsl(+/-) mice were viable, but the absence of cystatin M/E caused scarring alopecia in adult animals. In the cornea of Cst6(-/-)Ctsl(+/-) mice, we observed keratitis, hyperplasia, and transition to a cornified epithelium. Evidence is provided that activation of cathepsin D and transglutaminase-1 are downstream events, dependent of cathepsin L activity. We conclude that a tightly regulated balance between cathepsin L and cystatin M/E is essential for tissue integrity in epidermis, hair follicles, and corneal epithelium.

Published
2010

34. A comprehensive analysis of pattern recognition receptors in normal and inflamed human epidermis: upregulation of dectin-1 in psoriasis.

Author

Koning, H.D. de, Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Joosten, L.A.B., Netea, M.G., Schalkwijk, J., Zeeuwen, P.L.J.M., Koning, H.D. de, Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Joosten, L.A.B., Netea, M.G., Schalkwijk, J., and Zeeuwen, P.L.J.M.

Abstract

1 november 2010, Contains fulltext : 89335.pdf (publisher's version ) (Closed access), Human epidermis plays an important role in host defense by acting as a physical barrier and signaling interface between the environment and the immune system. Pattern recognition receptors (PRRs) are crucial to maintain homeostasis and provide protection during infection, but are also causally involved in monogenic auto-inflammatory diseases. This study aimed to investigate the epidermal expression of PRRs and several associated host defense molecules in healthy human skin, psoriasis, and atopic dermatitis (AD). Using microarray analysis and real-time quantitative PCR, we found that many of these genes are transcribed in normal human epidermis. Only a few genes were differentially induced in psoriasis (CLEC7A (dectin-1), Toll-like receptor (TLR) 4, and mannose receptor C type 1 (MRC1)) or AD (MRC1, IL1RN, and IL1beta) compared with normal epidermis. A remarkably high expression of dectin-1 mRNA was observed in psoriatic epidermis and this was corroborated by immunohistochemistry. In cultured primary human keratinocytes, dectin-1 expression was induced by IFN-gamma, IFN-alpha, and Th17 cytokines. Keratinocytes were unresponsive, however, to dectin-1 ligands such as beta-glucan or heat-killed Candida albicans, nor did we observe synergy with TLR2/TLR5 ligands. In conclusion, upregulation of dectin-1 in psoriatic lesions seems to be under control of psoriasis-associated cytokines. Its role in the biology of skin inflammation and infection remains to be explored.

Published
2010

35. Expression of the vanin gene family in normal and inflamed human skin: induction by proinflammatory cytokines.

Author

Jansen, P.A.M., Kamsteeg, M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Jongh, G.J. de, Bergers, M., Tjabringa, G.S., Zeeuwen, P.L.J.M., Schalkwijk, J., Jansen, P.A.M., Kamsteeg, M., Rodijk-Olthuis, D., Vlijmen-Willems, I.M.J.J. van, Jongh, G.J. de, Bergers, M., Tjabringa, G.S., Zeeuwen, P.L.J.M., and Schalkwijk, J.

Abstract

Contains fulltext : 80705.pdf (publisher's version ) (Closed access), The vanin gene family encodes secreted and membrane-bound ectoenzymes that convert pantetheine into pantothenic acid and cysteamine. Recent studies in a mouse colitis model indicated that vanin-1 has proinflammatory activity and suggest that pantetheinases are potential therapeutic targets in inflammatory diseases. In a microarray analysis of epidermal gene expression of psoriasis and atopic dermatitis lesions, we identified vanin-3 as the gene showing the highest differential expression of all annotated genes that we studied (19-fold upregulation in psoriasis). Quantitative real-time PCR analysis confirmed the microarray data on vanin-3 and showed similar induction of vanin-1, but not of vanin-2, in psoriatic epidermis. Immunohistochemistry showed that vanin-3 is expressed in the differentiated epidermal layers. Using submerged and organotypic keratinocyte cultures, we found that vanin-1 and vanin-3 are induced at the mRNA and protein level by psoriasis-associated proinflammatory cytokines (Th17/Th1) but not by Th2 cytokines. We hypothesize that increased levels of pantetheinase activity are part of the inflammatory-regenerative epidermal differentiation program, and may contribute to the phenotype observed in psoriasis.

Published
2009

36. Psoriasis is associated with increased beta-defensin genomic copy number.

Author

Hollox, E.J., Huffmeier, U., Zeeuwen, P.L.J.M., Palla, R., Lascorz, J., Rodijk-Olthuis, D., Kerkhof, P.C.M. van de, Traupe, H., Jongh, G.J. de, Heijer, M. den, Reis, A., Armour, J.A., Schalkwijk, J., Hollox, E.J., Huffmeier, U., Zeeuwen, P.L.J.M., Palla, R., Lascorz, J., Rodijk-Olthuis, D., Kerkhof, P.C.M. van de, Traupe, H., Jongh, G.J. de, Heijer, M. den, Reis, A., Armour, J.A., and Schalkwijk, J.

Abstract

Contains fulltext : 71309.pdf (publisher's version ) (Closed access), Psoriasis is a common inflammatory skin disease with a strong genetic component. We analyzed the genomic copy number polymorphism of the beta-defensin region on human chromosome 8 in 179 Dutch individuals with psoriasis and 272 controls and in 319 German individuals with psoriasis and 305 controls. Comparisons in both cohorts showed a significant association between higher genomic copy number for beta-defensin genes and risk of psoriasis.

Published
2008

37. Genetically programmed differences in epidermal host defense between psoriasis and atopic dermatitis patients

Author

Zeeuwen, P.L.J.M., Jongh, G.J. de, Rodijk-Olthuis, D., Kamsteeg, M., Verhoosel, R.M., Rossum, M.M. van, Hiemstra, P.S., Schalkwijk, J., Zeeuwen, P.L.J.M., Jongh, G.J. de, Rodijk-Olthuis, D., Kamsteeg, M., Verhoosel, R.M., Rossum, M.M. van, Hiemstra, P.S., and Schalkwijk, J.

Abstract

Contains fulltext : 70186.pdf (publisher's version ) (Open Access), In the past decades, chronic inflammatory diseases such as psoriasis, atopic dermatitis, asthma, Crohn's disease and celiac disease were generally regarded as immune-mediated conditions involving activated T-cells and proinflammatory cytokines produced by these cells. This paradigm has recently been challenged by the finding that mutations and polymorphisms in epithelium-expressed genes involved in physical barrier function or innate immunity, are risk factors of these conditions. We used a functional genomics approach to analyze cultured keratinocytes from patients with psoriasis or atopic dermatitis and healthy controls. First passage primary cells derived from non-lesional skin were stimulated with pro-inflammatory cytokines, and expression of a panel of 55 genes associated with epidermal differentiation and cutaneous inflammation was measured by quantitative PCR. A subset of these genes was analyzed at the protein level. Using cluster analysis and multivariate analysis of variance we identified groups of genes that were differentially expressed, and could, depending on the stimulus, provide a disease-specific gene expression signature. We found particularly large differences in expression levels of innate immunity genes between keratinocytes from psoriasis patients and atopic dermatitis patients. Our findings indicate that cell-autonomous differences exist between cultured keratinocytes of psoriasis and atopic dermatitis patients, which we interpret to be genetically determined. We hypothesize that polymorphisms of innate immunity genes both with signaling and effector functions are coadapted, each with balancing advantages and disadvantages. In the case of psoriasis, high expression levels of antimicrobial proteins genes putatively confer increased protection against microbial infection, but the biological cost could be a beneficial system gone awry, leading to overt inflammatory disease.

Published
2008

38. OR5-002 – In vitro studies in Schnitzler’s syndrome

Author

De Koning, HD, primary, Schalkwijk, J, additional, Jongekrijg, J, additional, van Vlijmen-Willems, IM, additional, Rodijk-Olthuis, D, additional, Stoffels, M, additional, van der Meer, JW, additional, Zeeuwen, PL, additional, and Simon, A, additional

Published
2013
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41. Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models.

Author

van den Brink NJM, Pardow F, Meesters LD, van Vlijmen-Willems I, Rodijk-Olthuis D, Niehues H, Jansen PAM, Roelofs SH, Brewer MG, van den Bogaard EH, and Smits JPH

Subjects
Humans, Cells, Cultured, Dermatitis, Atopic pathology, Dermatitis, Atopic metabolism, Psoriasis pathology, Cytokines metabolism, Electric Impedance, Epidermis metabolism, Keratinocytes physiology, Keratinocytes metabolism, Filaggrin Proteins, Dielectric Spectroscopy methods, Cell Differentiation
Abstract

Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EIS diff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EIS SC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EIS diff . Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EIS diff . This cytokine-associated decrease in EIS diff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2024
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42. The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A.

Author

Smits JPH, Qu J, Pardow F, van den Brink NJM, Rodijk-Olthuis D, van Vlijmen-Willems IMJJ, van Heeringen SJ, Zeeuwen PLJM, Schalkwijk J, Zhou H, and van den Bogaard EH

Subjects
Humans, Basic Helix-Loop-Helix Transcription Factors metabolism, Basic Helix-Loop-Helix Transcription Factors genetics, Signal Transduction, Cells, Cultured, Receptors, Aryl Hydrocarbon metabolism, Receptors, Aryl Hydrocarbon genetics, Transcription Factor AP-2 metabolism, Transcription Factor AP-2 genetics, Cell Differentiation, Keratinocytes metabolism, Keratinocytes cytology, Keratinocytes physiology, Filaggrin Proteins, Epidermis metabolism
Abstract

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as an indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multiomics analyses using human skin keratinocytes revealed that upon ligand activation, AHR binds open chromatin to induce expression of transcription factors, for example, TFAP2A, as a swift response to environmental stimuli. The terminal differentiation program, including upregulation of barrier genes, FLG and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2024
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43. CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

Author

Arnold KA, Moran MC, Shi H, van Vlijmen-Willems IMJJ, Rodijk-Olthuis D, Smits JPH, and Brewer MG

Subjects
Humans, Skin Diseases genetics, Skin Diseases metabolism, Tight Junctions metabolism, Keratin-10 metabolism, Keratin-10 genetics, Gene Knockout Techniques, Cell Proliferation, CRISPR-Cas Systems, Keratinocytes metabolism, Claudin-1 metabolism, Claudin-1 genetics, Filaggrin Proteins metabolism, Cell Differentiation, Epidermis metabolism, Epidermis pathology
Abstract

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Published
2024
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44. Novel methodologies for host-microbe interactions and microbiome-targeted therapeutics in 3D organotypic skin models.

Author

Rikken G, Meesters LD, Jansen PAM, Rodijk-Olthuis D, van Vlijmen-Willems IMJJ, Niehues H, Smits JPH, Oláh P, Homey B, Schalkwijk J, Zeeuwen PLJM, and van den Bogaard EH

Subjects
Animals, Humans, Skin microbiology, Epidermis, Models, Animal, Host Microbial Interactions, Microbiota
Abstract

Background: Following descriptive studies on skin microbiota in health and disease, mechanistic studies on the interplay between skin and microbes are on the rise, for which experimental models are in great demand. Here, we present a novel methodology for microbial colonization of organotypic skin and analysis thereof., Results: An inoculation device ensured a standardized application area on the stratum corneum and a homogenous distribution of bacteria, while preventing infection of the basolateral culture medium even during prolonged culture periods for up to 2 weeks at a specific culture temperature and humidity. Hereby, host-microbe interactions and antibiotic interventions could be studied, revealing diverse host responses to various skin-related bacteria and pathogens., Conclusions: Our methodology is easily transferable to a wide variety of organotypic skin or mucosal models and different microbes at every cell culture facility at low costs. We envision that this study will kick-start skin microbiome studies using human organotypic skin cultures, providing a powerful alternative to experimental animal models in pre-clinical research. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Published
2023
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45. CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope Proteins.

Author

Niehues H, Rikken G, Kersten FFJ, Eeftens JM, van Vlijmen-Willems IMJJ, Rodijk-Olthuis D, Jansen PAM, Hendriks WJAJ, Ederveen THA, Schalkwijk J, van den Bogaard EH, and Zeeuwen PLJM

Subjects
Cornified Envelope Proline-Rich Proteins genetics, Cornified Envelope Proline-Rich Proteins metabolism, Epidermis metabolism, Keratinocytes metabolism, Proteins metabolism, Skin metabolism, Humans, Anti-Infective Agents metabolism, Psoriasis genetics, Psoriasis metabolism
Abstract

Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced in vivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2023
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46. Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes.

Author

Smits JPH, van den Brink NJM, Meesters LD, Hamdaoui H, Niehues H, Jansen PAM, van Vlijmen-Willems IMJJ, Rodijk-Olthuis D, Evrard C, Poumay Y, van Geel M, Hendriks WJAJ, Schalkwijk J, Zeeuwen PLJM, and van den Bogaard EH

Subjects
Humans, Filaggrin Proteins, Keratinocytes metabolism, Phenotype, CRISPR-Cas Systems, Intermediate Filament Proteins metabolism
Abstract

Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2023
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47. The aryl hydrocarbon receptor regulates epidermal differentiation through transient activation of TFAP2A.

Author

Smits JPH, Qu J, Pardow F, van den Brink NJM, Rodijk-Olthuis D, van Vlijmen-Willems IMJJ, van Heeringen SJ, Zeeuwen PLJM, Schalkwijk J, Zhou H, and van den Bogaard EH

Abstract

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases., Competing Interests: Conflict of interest The authors declare no conflicts of competing financial interests.

Published
2023
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48. Gram-positive anaerobic cocci guard skin homeostasis by regulating host-defense mechanisms.

Author

van der Krieken DA, Rikken G, Ederveen THA, Jansen PAM, Rodijk-Olthuis D, Meesters LD, van Vlijmen-Willems IMJJ, van Cranenbroek B, van der Molen RG, Schalkwijk J, van den Bogaard EH, and Zeeuwen PLJM

Abstract

In atopic dermatitis (AD), chronic skin inflammation is associated with skin barrier defects and skin microbiome dysbiosis including a lower abundance of Gram-positive anaerobic cocci (GPACs). We here report that, through secreted soluble factors, GPAC rapidly and directly induced epidermal host-defense molecules in cultured human keratinocytes and indirectly via immune-cell activation and cytokines derived thereof. Host-derived antimicrobial peptides known to limit the growth of Staphylococcus aureus -a skin pathogen involved in AD pathology-were strongly upregulated by GPAC-induced signaling through aryl hydrocarbon receptor (AHR)-independent mechanisms, with a concomitant AHR-dependent induction of epidermal differentiation genes and control of pro-inflammatory gene expression in organotypic human epidermis. By these modes of operandi, GPAC may act as an "alarm signal" and protect the skin from pathogenic colonization and infection in the event of skin barrier disruption. Fostering growth or survival of GPAC may be starting point for microbiome-targeted therapeutics in AD., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Published
2023
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49. Identification of Keratinocyte Mitogens: Implications for Hyperproliferation in Psoriasis and Atopic Dermatitis.

Author

Niehues H, Rikken G, van Vlijmen-Willems IMJJ, Rodijk-Olthuis D, van Erp PEJ, Zeeuwen PLJM, Schalkwijk J, and van den Bogaard EH

Abstract

Psoriasis and atopic dermatitis are chronic inflammatory skin diseases characterized by keratinocyte (KC) hyperproliferation and epidermal acanthosis (hyperplasia). The milieu of disease-associated cytokines and soluble factors is considered a mitogenic factor; however, pinpointing the exact mitogens in this complex microenvironment is challenging. We employed organotypic human epidermal equivalents, faithfully mimicking native epidermal proliferation and stratification, to evaluate the proliferative effects of a broad panel of (literature-based) potential mitogens. The KC GF molecule, the T-helper 2 cytokines IL-4 and IL-13, and the psoriasis-associated cytokine IL-17A caused acanthosis by hyperplasia through a doubling in the number of proliferating KCs. In contrast, IFN-γ lowered proliferation, whereas IL-6, IL-20, IL-22, and oncostatin M induced acanthosis not by hyperproliferation but by hypertrophy. The T-helper 2‒cytokine‒mediated hyperproliferation was Jak/signal transducer and activator of transcription 3 dependent, whereas IL-17A and KC GF induced MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase‒dependent proliferation. This discovery that key regulators in atopic dermatitis and psoriasis are direct KC mitogens not only adds evidence to their crucial role in the pathophysiological processes but also highlights an additional therapeutic pillar for the mode of action of targeting biologicals (e.g., dupilumab) or small-molecule drugs (e.g., tofacitinib) by the normalization of KC turnover within the epidermal compartment., (© 2021 The Authors.)

Published
2021
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50. Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures.

Author

Niehues H, Jansen PAM, Rodijk-Olthuis D, Rikken G, Smits JPH, Schalkwijk J, Zeeuwen PLJM, and van den Bogaard EHJ

Subjects
Adenoids cytology, Adenoids virology, DNA, Bacterial analysis, DNA, Viral analysis, Decontamination methods, Epithelial Cells, Equipment Contamination, Humans, Sanitary Engineering, Sequence Analysis, DNA, Water Microbiology, Adenoviruses, Human isolation & purification, Brevibacillus isolation & purification, Primary Cell Culture
Abstract

In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination., (© 2020 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Published
2020
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